Potential endocrine correlation with exposure to domoic acid in Southern Right Whale (Eubalaena australis) at the Península Valdés breeding ground.

In waters off Península Valdés (PV), Argentina, southern right whales (SRW, Eubalaena australis) are occasionally exposed to domoic acid (DA), a neurotoxin produced by diatoms of the genus Pseudo-nitzschia. Domoic acid toxicity in marine mammals can cause gastrointestinal and neurological clinical signs, alterations in hematologic and endocrine variables, and can be fatal in extreme cases. In this study, we validated an enzyme immunoassay to quantify fecal glucocorticoid metabolites (fGCm) in 16 SRW fecal samples from live and dead stranded whales in PV from 2013 to 2018 and assessed fGCm levels associated with DA exposure. Overall, fGCm levels were significantly lower in SRWs with detectable fecal DA (n = 3) as compared to SRWs with undetectable fecal DA levels (n = 13). The highest fecal DA was observed in a live lactating female, which had low fGCm compared to the other lactating females studied. The highest fGCm was observed in a lactating female with undetectable DA; interestingly, at the time of sample collection, this female was sighted with two calves, an extremely unusual occurrence in this species. Though the sample size of these exceptionally rare breeding-season fecal samples was unavoidably small, our study provides evidence of potential adrenal alterations in whales exposed to an environmental neurotoxin such as DA.

Molecularly imprinted nanoparticle-based assay (MINA): Potential application for the detection of the neurotoxin domoic acid.

Domoic acid (DA) is a natural amino acid and water-soluble neurotoxic biotoxin primarily produced by the microalgae Pseudo-nitzschia. DA can cause poisoning in humans and a wide variety of marine species. In this work, a molecularly imprinted nanoparticle-based assay (MINA) was developed as an alternative to enzyme-linked immunosorbent assay (ELISA) for selective detection of DA. In contrast with ELISA, MINA uses molecularly imprinted polymer nanoparticles (nanoMIPs) as plastic antibodies due to its higher stability and lower production costs. In this work, dihydrokainic acid (DKA) was used as a dummy template because this molecule is structurally similar to DA but less toxic. The developed MINA had a high linear response for DKA and DA, showing detection limits of 2.12 nmol L-1 and 4.32 nmol L-1, respectively. Additionally, q-RMN studies demonstrated that DKA - nanoMIPs were selective for DKA, since they presented the best association parameters with a high loading load capacity of 175% and an association efficiency of 18%. No cross-reactivity towards 1, 3, 5 - pentanetricarboxylic acid was observed. These results suggest that MINA could be a more robust, more sensitive, and less expensive alternative to ELISA. The assay developed with DKA - nanoMIPs has strong potential for the detection of domoic acid in real samples of red tide.

First Report of Domoic Acid Production from Pseudo-nitzschia multistriata in Paracas Bay (Peru).

The Peruvian sea is one of the most productive ecosystems in the world. Phytoplankton production provides food for fish, mammals, mollusks and birds. This trophic network is affected by the presence of toxic phytoplankton species. In July 2017, samples of phytoplankton were obtained from Paracas Bay, an important zone for scallop (Argopecten purpuratus) aquaculture in Peru. Morphological analysis revealed the presence of the genus Pseudo-nitzschia, which was isolated and cultivated in laboratory conditions. Subsequently, the monoclonal cultures were observed by scanning electron microscopy (SEM), and identified as P. multistriata, based on both the morphological characteristics, and internal transcribed spacers region (ITS2) sequence phylogenetic analysis. Toxin analysis using liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) revealed the presence of domoic acid (DA) with an estimated amount of 0.004 to 0.010 pg cell-1. This is the first report of DA from the coastal waters of Peru and its detection in P. multistriata indicates that it is a potential risk. Based on our results, routine monitoring of this genus should be considered in order to ensure public health.

Rapid Domoic Acid Depuration in the Scallop Argopecten purpuratus and Its Transfer from the Digestive Gland to Other Organs.

Domoic acid (DA), the main toxin responsible for Amnesic Shellfish Poisoning, frequently affects the marine resources of Chile and other countries across the South Pacific, thus becoming a risk for human health. One of the affected resources is the scallop Argopecten purpuratus. Even though this species has a high commercial importance in Northern Chile and Peru, the characteristics of its DA depuration are not known. In this work, the DA depuration was studied by means of two experiments: one in controlled (laboratory) and another in natural conditions. All organs of A. purpuratus depurated the toxin very quickly in both experiments. In some organs, an increase or a very small decrease of toxin was detected in the early depuration steps. Several models were used to describe this kinetics. The one that included toxin transfer between organs and independent depuration from each organ was the model that best fit the data. It seems, therefore, that the DA in this species is quickly transferred from the digestive gland to all other organs, which release it into the environment. Physiological differences in the two experiments have been shown to have some effect on the depuration from each organ but the actual reasons are still unknown.

Toxigenic phytoplankton groups and neurotoxin levels related to two contrasting environmental conditions at the coastal area of Rio de Janeiro (west of South Atlantic).

An assessment of the major pigments and neurotoxins and a description of the phytoplankton community were carried out within the coastal region of Rio de Janeiro State (Brazil), during winter and the following spring of 2018. Overall, six stations were investigated for oceanographic conditions (with CTD casts). Filtered water samples were used to estimate the chlorophyll a (CHL-a), carotenoids (CAR), and phycobiliproteins (PHY) using UV-Vis spectrophotometry, as well as the quantification of saxitoxins (STX) and domoic acid (DA), through High Performance Liquid Chromatography (HPLC). Planktonic organisms were counted using sedimentation chambers of different volumes and an inverted microscope. A cluster analysis, SIMPER, and ANOSIM were applied to the phytoplankton data along with diversity indexes, and non-parametric statistics to phycotoxins and pigments. There was a significant difference between the winter and spring phytoplankton community, associated with the mixed layer depth (r2 = -0.626, p < 0.05) and temperature (r2 = 0.641, p < 0.05). Phytoplankton biomass and C:CHL-a indicated a higher production during the winter than in spring, with the potentially toxic genus Pseudo-nitzschia responsible for 12.79% of autotrophic abundance (SIMPER output). Pigments showed a slight increase in CAR during spring, while PHY remained at trace concentrations. Both the DA and STX were quantified in winter and spring, but with significant differences only for STX between the sampling periods. Among the 71 taxa, 11 were identified as potentially toxic with an emphasis on STX-producing dinoflagellates and cyanobacteria, such as Alexandrium sp., Gymnodinium spp. along with Trichodesmium spp. Season-related environmental variability may be the major driving force modulating the mixed assemblage of species that support different levels of phycotoxins.

Phylogenetic relationships of Pseudo-nitzschia subpacifica (Bacillariophyceae) from the Mexican Pacific, and its production of domoic acid in culture.

Pseudo-nitzschia is a cosmopolitan genus, some species of which can produce domoic acid (DA), a neurotoxin responsible for the Amnesic Shellfish Poisoning (ASP). In this study, we identified P. subpacifica for the first time in Todos Santos Bay and Manzanillo Bay, in the Mexican Pacific using SEM and molecular methods. Isolates from Todos Santos Bay were cultivated under conditions of phosphate sufficiency and deficiency at 16°C and 22°C to evaluate the production of DA. This toxin was detected in the particulate (DAp) and dissolved (DAd) fractions of the cultures during the exponential and stationary phases of growth of the cultures. The highest DA concentration was detected during the exponential phase grown in cells maintained in P-deficient medium at 16°C (1.14 ± 0.08 ng mL-1 DAd and 4.71 ± 1.11 × 10-5 ng cell-1 of DAp). In P-sufficient cultures DA was higher in cells maintained at 16°C (0.25 ± 0.05 ng mL-1 DAd and 9.41 ± 1.23 × 10-7 ng cell-1 of DAp) than in cells cultured at 22°C. Therefore, we confirm that P. subpacifica can produce DA, especially under P-limited conditions that could be associated with extraordinary oceanographic events such as the 2013-2016 "Blob" in the northeastern Pacific Ocean. This event altered local oceanographic conditions and possibly generated the presence of potential harmful species in areas with economic importance on the Mexican Pacific coast.

Occurrence of Toxigenic Microalgal Species and Phycotoxin Accumulation in Mesozooplankton in Northern Patagonian Gulfs, Argentina.

In the Northern Patagonian gulfs of Argentina (Golfo Nuevo and Golfo San José), blooms of toxigenic microalgae and the detection of their associated phycotoxins are recurrent phenomena. The present study evaluated the transfer of phycotoxins from toxigenic microalgae to mesozooplankton in Golfo Nuevo and Golfo San José throughout an annual cycle (December 2014-2015 and January 2015-2016, respectively). In addition, solid-phase adsorption toxin tracking (SPATT) samplers were deployed for the first time in these gulfs, to estimate the occurrence of phycotoxins in the seawater between the phytoplankton samplings. Domoic acid was present throughout the annual cycle in SPATT samplers, whereas no paralytic shellfish poisoning toxins were detected. Ten toxigenic species were identified: Alexandrium catenella, Dinophysis acuminata, Dinophysis acuta, Dinophysis tripos, Dinophysis caudata, Prorocentrum lima, Pseudo-nitzschia australis, Pseudo-nitzschia calliantha, Pseudo-nitzschia fraudulenta, and Pseudo-nitzschia pungens. Lipophilic and hydrophilic toxins were detected in phytoplankton and mesozooplankton from both gulfs. Pseudo-nitzschia spp. were the toxigenic species most frequent in these gulfs. Consequently, domoic acid was the phycotoxin most abundantly detected and transferred to upper trophic levels. Spirolides were detected in phytoplankton and mesozooplankton for the first time in the study area. Likewise, dinophysistoxins were found in mesozooplankton from both gulfs, and this is the first report of the presence of these phycotoxins in zooplankton from the Argentine Sea. The dominance of calanoid copepods indicates that they were the primary vector of phycotoxins in the pelagic trophic web. Environ Toxicol Chem 2019;38:2209-2223. © 2019 SETAC.

Domoic acid in the tropical South Atlantic Ocean - An environment case study.

Domoic acid (DA) or Amnesic Shellfish Poisoning (ASP) produced by the genus Pseudo-nitzschia diatom was investigated in two seasonal periods in fishing areas of Katsuwonus pelamis in the South Atlantic Ocean. Higher DA concentrations were found in spring compared to winter. Pseudo-nitzschia spp. more quantified in winter than in spring, while P. pungens, a species among the most reported for an AD toxic potential, was only found in spring.

The polychaete, Paraprionospio pinnata, is a likely vector of domoic acid to the benthic food web in the northern Gulf of Mexico.

A somewhat disparate, yet temporally cohesive, set of phytoplankton abundance, microphytobenthos, including the diatom Pseudo-nitzschia, benthic infauna, and sediment toxin data were used to develop a theory for the transfer of domoic acid (DA) from the toxic diatom to the benthos in the highly productive waters of the northern Gulf of Mexico near the Mississippi River plume. Archived samples and new data were used to test the theory that DA is likely to be incorporated into benthic consumers. High spring abundances of potentially toxic Pseudo-nitzschia diatoms were simultaneously present in the surface waters, bottom waters and on the seafloor. Examination of the gut contents of a typical deposit-feeding and suspension-feeding polychaete, Paraprionospio pinnata, during similar periods of high Pseudo-nitzschia abundance in surface water indicated consumption of the diatoms. Demersal fishes, particularly Atlantic croaker, are known to consume these polychaetes, with a potential for transfer of DA to even higher trophic levels. These findings warrant a theory to be tested with further studies about the trophic linkage of a phytoplankton toxin into the benthic food web.

Growth, chain formation, and toxin production by southern Brazilian Pseudo-nitzschia isolates under laboratory conditions.

Pseudo-nitzschia is a diatom genus capable of producing the neurotoxin domoic acid (DA), which has been related to mortalities of marine vertebrates, and the amnesic shellfish poisoning (ASP) in human consumers of contaminated bivalves. This study reports DA production among Pseudo-nitzschia strains isolated from shellfish farming areas in southern Brazil. Twenty-seven cultures of potentially toxigenic Pseudo-nitzschia species were established. Growth, stepped-chain formation, and DA production were evaluated in static, intermittently illuminated (12:12 photoperiod) batch cultures for 12 selected strains, and under continuous light and/or turbulence for a single Pseudo-nitzschia calliantha strain. Growth rate ranged from 0.16 to 0.39 day-1 among the 12 strains. Only P. calliantha and P. cf. multiseries yielded detectable levels of intracellular DA, reaching up to 0.054 fg cell-1 in late exponential and 0.15 pg cell-1 in early stationary phase, respectively. Continuous light impaired cell growth, and turbulence enhanced step-chain formation by threefold during exponential growth phase, but no DA production was detected under both conditions. The effect of turbulence on chain formation should be further evaluated in the field, once particle size is expected to affect the ingestion of toxic cells and thus toxin accumulation by certain DA vectors. The low cell toxicity revealed herein under laboratory conditions is in accordance with the low frequency of DA contamination episodes in south Brazilian waters. Nevertheless, monitoring of Pseudo-nitzschia abundance and DA presence in farming areas is continuously required to assure the quality of local shellfish products.

Growth, Toxin Production and Allelopathic Effects of Pseudo-nitzschia multiseries under Iron-Enriched Conditions.

In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries, static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (µ = 0.45-0.73 d-1) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2-4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50-70% reduction in cell density and 70-90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species.

Domoic acid in a marine pelagic food web: Exposure of southern right whales Eubalaena australis to domoic acid on the Península Valdés calving ground, Argentina.

The gulfs that surround Península Valdés (PV), Golfo Nuevo and Golfo San José in Argentina, are important calving grounds for the southern right whale Eubalaena australis. However, high calf mortality events in recent years could be associated with phycotoxin exposure. The present study evaluated the transfer of domoic acid (DA) from Pseudo-nitzschia spp., potential producers of DA, to living and dead right whales via zooplanktonic vectors, while the whales are on their calving ground at PV. Phytoplankton and mesozooplankton (primary prey of the right whales at PV and potential grazers of Pseudo-nitzschia cells) were collected during the 2015 whale season and analyzed for species composition and abundance. DA was measured in plankton and fecal whale samples (collected during whale seasons 2013, 2014 and 2015) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The genus Pseudo-nitzschia was present in both gulfs with abundances ranging from 4.4×102 and 4.56×105 cell l-1. Pseudo-nitzschia australis had the highest abundance with up to 4.56×105 cell l-1. DA in phytoplankton was generally low, with the exception of samples collected during a P. australis bloom. No clear correlation was found between DA in phytoplankton and mesozooplankton samples. The predominance of copepods in mesozooplankton samples indicates that they were the primary vector for the transfer of DA from Pseudo-nitzschia spp. to higher trophic levels. High levels of DA were detected in four whale fecal samples (ranging from 0.30 to 710µgg-1 dry weight of fecal sample or from 0.05 and 113.6µgg-1 wet weight assuming a mean water content of 84%). The maximum level of DA detected in fecal samples (710µg DA g-1 dry weight of fecal sample) is the highest reported in southern right whales to date. The current findings demonstrate for the first time that southern right whales, E. australis, are exposed to DA via copepods as vectors during their calving season in the gulfs of PV.

Species occurrence of the potentially toxigenic diatom genus Pseudo-nitzschia and the associated neurotoxin domoic acid in the Argentine Sea.

The marine diatom genus Pseudo-nitzschia, the major known producer of the neurotoxin domoic acid (DA) responsible for the amnesic shellfish poisoning (ASP) syndrome in humans and marine mammals, is globally distributed. The genus presents high species richness in the Argentine Sea and DA has been frequently detected in the last few years in plankton and shellfish samples, but the species identity of the producers remains unclear. In the present work, the distribution and abundance of Pseudo-nitzschia species and DA were determined from samples collected on two oceanographic cruises carried out through the Argentine Sea (∼39-47°S) during summer and spring 2013. Phytoplankton composition was analysed by light and electron microscopy while DA was determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The genus Pseudo-nitzschia was recorded in 71 and 86% of samples collected in summer and spring, respectively, whereas DA was detected in only 42 and 21% of samples, respectively. Microscopic analyses revealed at least five potentially toxic species (P. australis, P. brasiliana, P. fraudulenta, P. pungens, P. turgidula), plus putatively non-toxigenic P. dolorosa, P. lineola, P. turgiduloides and unidentified specimens of the P. pseudodelicatissima complex. The species P. australis showed the highest correlation with DA occurrence (r=0.55; p<0.05), suggesting its importance as a major DA producer in the Argentine Sea. In the northern area and during summer, DA was associated with the presence of P. brasiliana, a species recorded for the first time in the Argentine Sea. By contrast, high concentrations of P. fraudulenta, P. pungens and P. turgidula did not correspond with DA occurrence. This study represents the first successful attempt to link toxigenicity with Pseudo-nitzschia diversity and cell abundance in field plankton populations in the south-western Atlantic.

Depuration and anatomical distribution of domoic acid in the surf clam Mesodesma donacium.

In northern Chile, domoic acid (DA) has been detected in several bivalve species. In Mesodesma donacium, one of the most important commercial species for local fishermen, no information is available on depuration, or on the anatomical distribution of this toxin and its potential use as a palliative measure to minimize the consequences of ASP outbreaks. Deputation of DA is very fast in M. donacium, and can be adequately described by means of a two-compartment model. The estimated rates for the first and second compartments were 1.27 d(-1) and 0.24 d(-1), respectively, with a transfer rate between compartments of 0.75. Having high depuration rates protects this species from being affected by Pseudo-nitzschia blooms for an extended period of time. Taking this into account, the time in which the bivalves are unsafe for consumers is very short, and therefore the economic losses that could result by the DA outbreaks in local fisheries should be moderate. In relation to anatomical distribution, at least during the uptake phase, the toxin was evenly distributed within the soft tissues, with a total toxin burden corresponding to 27%, 32% and 41% for Digestive Gland (DG), Foot (FT) and Other Body Fractions (OBF), respectively. Since the contribution of each organ to the toxin concentration is a function of both weight contribution and toxin burden, the pattern of toxin distribution showed the following trend: "all other body fractions" (OBF) > Foot (FT) > Digestive Gland (DG). Thus, the highest concentration of DA, with a contribution close to 72%, corresponds to the edible tissues (OBF + FT), while the DG (non-edible tissue) only contributes the remaining 28%. Consequently, in view of the anatomical distribution of domoic acid in M. donacium, the elimination of the digestive gland does not substantially reduce the toxicity of the final product and therefore selective evisceration would not improve their quality for human consumption.

The occurrence of domoic acid linked to a toxic diatom bloom in a new potential vector: the tunicate Pyura chilensis (piure).

The tunicate Pyura chilensis (Molina, 1782); Phylum Chordata; Subphylum Urochordata; Class Ascidiacea, common local name "piure" or sea squirt; a filter-feeder (plankton and suspended particles) sessile species; may play an important role in monitoring domoic acid (DA) the principal toxic component of Amnesic Shellfish Poisoning (ASP). Significant DA concentrations have been determined in tunicate samples, collected during a recent ASP outbreak in Bahía Inglesa, an important scallop (Argopecten purpuratus) farming area. Several infaunal species were tested for the presence of DA, in addition to the usual scallop monitoring programme. DA was found at sub-toxic levels in filtering bivalves such as mussels (Mytilus chilensis), large mussels (Aulacomya ater) and clams (Protothaca thaca) (6.4, 5.4 and 4.7 microg DA/g tissue respectively). Of particular interest was the observation of significant accumulations of toxic Pseudo-nitzschia sp. diatoms in the internal siphon and atrium spaces of the tunicate. Toxin distribution within major tunicate organs was heterogeneous with 8.7-15.5 microg DA/g in edible tissues, 14.9-17.9 microg DA/g in the fecal material and 13.6-32.7 microg DA/g in the gut content. DA was determined by HPLC-UV and confirmed by diode-array detection and LC-MS/MS analysis. This is the first report of the presence of DA in a tunicate that is regularly consumed by coastal populations. These results confirm the need to include these organisms in sanitation programs for marine toxins.

Evidence that glutaric acid reduces glutamate uptake by cerebral cortex of infant rats.

The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.

Comparative study of Domoic Acid and Okadaic Acid induced-chromosomal abnormalities in the Caco-2 cell line.

Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.

Behavioral and histopathological analysis of domoic Acid administration in marmosets.

PURPOSE: To induce status epilepticus (SE) followed by the subsequent onset of spontaneous recurrent seizures, thus characterizing a new model of temporal lobe epilepsy in a nonhuman primate. METHODS: Male and female marmosets (Callithrix jacchus) (n = 18), ages between 2 and 8 years, were injected with domoic acid (0.5-4 mg/kg, i.p.) or saline, and behaviorally assessed with regard to the presence of acutely induced seizures and for < or = 6 months for spontaneous seizures. Injection of doses ranging from 3.5 to 4 mg/kg either did not induce SE or resulted in fatal SE. Even a 5-min SE duration (SE blockade resulting from diazepam injection) proved lethal to marmosets within 1 h of domoate administration, regardless of intensive care and monitoring of the animals. Animals injected with doses ranging from 0.5 to 3 mg/kg that developed only a few minor convulsive signs were allowed a 6-month survival period for the assessment of spontaneous epileptic events. At the end of the experiment, 6-month period, or acute intoxication associated with SE induction, animals were deeply anesthetized and had their brains subjected to histologic processing for Nissl and delta-FosB. RESULTS: For the animals injected with domoate that did not develop SE (i.e., those that survived), we could not detect any behavioral signs of spontaneous epileptic seizures in the 6-month observation period, and only minor indications of neuropathologic changes (i.e., neuronal death) over Nissl-stained sections, as well as some small changes in the staining for delta-FosB in a few of the animals. CONCLUSIONS: Systemic administration of domoic acid to marmosets is not effective for the generation of a model of chronic temporal lobe epilepsy. Administration of domoic acid at doses that do not lead to SE also did not lead to the development of temporal lobe epilepsy or clear-cut behavioral changes over a 6-month period.

Improved high-performance liquid chromatographic method for the determination of domoic acid and analogues in shellfish: effect of pH.

Domoic acid (DA) is a naturally-occurring amino acid that causes a form of human intoxication called amnesic shellfish poisoning (ASP) following the consumption of shellfish. A rapid and sensitive HPLC-UV method has been developed for analysis of DA and analogues in shellfish without the need for SPE clean-up. Isocratic chromatographic separation of DA and its isomers from shellfish matrix interferences and from the prevalent amino acid, tryptophan, was achieved by careful control of the mobile phase pH. The optimised pH was found to be 2.5 when using a Luna(2) C18 column. Sample extraction was verified with control extracts from shellfish spiked at 5.0 and 10.0 microg/g of DA and with certified reference material. The average extraction efficiency was 98.5%. The calibration, based on mussel tissue spiked with DA standard, was linear in the range 0.05-5.0 microg/ml (r = 0.9999) and the detection limit (signal:noise 3:1) was better than 25 ng/ml. The DA assay achieved good precision; %RSD = 1.63 (intra-day, n = 6) and %RSD = 3.7 (inter-day, n = 8). This method was successfully applied to a variety of shellfish species, allowing the rapid screening of a large number of samples per day (20-30), without the need for SPE clean-up. Quantitative data were obtained for shellfish samples containing domoic acid in the concentration range 0.25-330 microg/g. Using the same chromatographic conditions, LC-MS3 was used to determine DA and its isomers, isodomoic acid D and epi-domoic acid, in scallop tissues.

Proliferaciones algales de la diatomea toxigénica Pseudo-Nitzschia (Bacillariophyceae) en el Golfo de Nicoya, Costa Rica / Algal blooms of the toxigenic diatom Pseudo-Nitzschia (Bacillariophyceae) in the Golfo de Nicoya, Costa Rica

En el mes de noviembre de 2001, se aisló de una marea roja cerca de la Isla San Lucas, las diatomeas Pseudo-Nitzschia pungens f. pungens seguida por Skeletonema costatum, Chaetoceros lorenzianus y en menor concentración Thalassiosira spp. las cuales fueron caracterizadas por microscopía electrónica de rastreo y transmisión, siendo esta la primera vez que se describe la presencia de estas especies produciendo proliferaciones en Costa Rica. Actualmente existe un aumento en el reporte de mareas rojas donde predominan las diatomeas, y los conteos celulares indican el aumento en el número de las especies conocidas como Pseudo-nitzschia pungens f. multiseries. A estas se les atribuye la producción del ácido domóico, un aminoácido de bajo peso molecular. Las intoxicaciones amnésicas por mariscos (IAM) que afectan a los humanos, se producen por la ingesta de mariscos contaminados con este ácido. En Costa Rica, hasta la fecha, solo se han reportado casos de intoxicación paralítica por mariscos contaminados por los dinoflagelados Pyrodinium bahamense var. compressum y Gymnodinium catenatum, sin embargo existe la posibilidad de producirse intoxicaciones humanas por la ingesta de mariscos y peces contaminados con toxinas amnésicas las cuales son hidrosolubles y termoestables. Debe entonces considerarse el peligro potencial de aparición de casos de intoxicaciones amnésicas y se sugiere la inclusión de estas especies en los programas de monitoreo permanente para tomar las medidas preventivas de salud pública

Water samples were collected during a red tide event in November 2001, near San Lucas Island (Gulf of Nicoya, Costa Rica). Superficial temperature was 27ºC and water was turbid, with no fetid smell. One sample was treated with negative staining and observed using a transmission electron microscope (TEM); another sample was observed using a scanning electron microscope (SEM). Samples had high concentrations of the diatom Pseudo-Nitzschia pungens f. pungens (characterized by two rows of poroids in the external channel), and lower concentrations of Skeletonema costatum (chains joined by external microtubules) and Chaetoceros lorenzianus (oval apertures and long chains, having setae with distinctive transverse rows and spines). This is the first time that the first species was described producing red tides in Costa Rica. However, reports about red tides with high concentration of species like P. pungens (variety multiseries) are increasing. These species have been related to the production of domoic acid, a low molecular weight amino acid which in humans can cause amnesic intoxications with seafood. Previously, Costa Rican reports of toxic accidents only referred to seafood contaminated with Pyrodinium bahamense var. compressum and Gymnodinium catenatum dinoflagellates. The increase in the number of Pseudo-Nitzschia causing harmful algae blooms is of interest for scientists around the world and must be documented. Similarly, some Chaetoceros species have been reported to be harmful to fish. We strongly recommend the establishment of a permanent surveillance program monitoring the presence of these species new at Costa Rican Pacific coast. Since the amnesic toxin is soluble in water and heat-resistant, we want to stress the possibility of having human cases of amnesic intoxication